1. Field of the Invention
The present invention relates to beta-glucosidase that is mutated to have enhanced activity, and a method for producing bioethanol using the same. More particularly, the present invention relates to a polynucleotide encoding beta-glucosidase that is mutated to have enhanced activity, beta-glucosidase expressed from the polynucleotide, an expression vector including the polynucleotide, a transformant that is transformed with the expression vector, a method for producing the mutated beta-glucosidase using the transformant, and a method for producing bioethanol using the transformant.
2. Description of the Related Art
Worldwide, efforts are ongoing to obtain the bio-energy from inexpensive, renewable biomass because of the problems of the depletion of crude oil and global warming. Cellulosic biomass is the most abundant organic material on Earth, and is a renewable raw material capable of producing a variety of energy and raw material platform compounds, which have been conventionally produced from the petroleum-based materials (Hoffer, et al., 2002, Science, 298, 981). The process of obtaining bio-energy, particularly, bio-ethanol from cellulosic biomass is technically possible, but it is expensive and lacks important benefits considering the current crude oil price (Zaldivar et al., 2001, Appl. Microbiol. Biotechnol., 56, 17). Bioethanol is obtained from cellulosic biomass by decomposition of biomass and fermentation of the decomposed sugar, but naturally occurring microorganisms cannot decompose and ferment biomass in a high yield at the same time. Inefficiently, the current technology consists of two steps, decomposition (saccharification) and fermentation of biomass (Lynd et al., 2002, Microbiol. Mol. Biol. Rev. 66, 506).
Because cellulosic biomass has a very solid structure and its natural decomposition occurs very slowly, it is required to perform pretreatment and expensive cellulase treatment processes in order to artificially speed up the decomposition (Lynd et al., 1999, Biotechnol. Prog. 15, 777, Himmel et al., 2007, Science, 315, 804). Therefore, in order to ensure economic feasibility in the production of bioenergy and platform compounds using cellulose biomass, it is required to develop a low-cost mass production technology of cellulases for efficient decomposition of cellulose biomass, in particular, a consolidated bioprocess (CBP) technology of directly utilizing the enzyme-producing recombinant strain in the bioenergy production technology (Hahn-Hagerdal et al., 2006, Trends Biotechnol, 24, 549, Lynd et al., 2008, Nat. Biotechnol., 26, 169).
Cellulosic biomass has a very solid and stable structure, because it is mainly composed of cellulose as a glucose polymer, hemicellulose as a xylose polymer, and lignin. For efficient enzymatic decomposition of cellulosic biomass, it is necessary to perform a physicochemical pretreatment of the biomass to disintegrate the stable structure of the plant, thereby allowing better access to substrate by the enzyme. A number of different types of cellulases are necessary, depending on the type of the substrate. For degradation of cellulose, endocellulase (endo-1,4-β-D-glucanase), exocellulase (exo-1,4-β-D-glucanase or cellobiohydrolase) and beta-glucosidase (β-glucosidase or cellobiase) are essential (Kubicek et al., 1992, Adv. Biochem. Eng. Biotechnol. 45, 1). For degradation of hemicellulose, endoxylanase (endo-1,4-β-xylanase) and beta-xylosidase (β-xylosidase) are needed as representative essential enzymes, and a variety of de-branching enzymes are required for complete degradation of cellulosic biomass. These enzymes are found in the microorganisms, in particular, fungi which naturally degrade plants. A commercially available cellulase is an enzyme complex derived from the fungus Trichoderma reesei, which has been commercialized by Novozymes and Danisco.
At present, the biomass-degrading enzyme for bioenergy production has been exclusively produced and sold by the two aforementioned multinational corporations in the world. However, the enzyme is considerably expensive, and it is not optimized for the type of biomass. Thus, there is a problem that an excessive amount of enzyme should be used for complete decomposition of cellulosic biomass (Merino and Cherry, 2007, Adv. Biochem. Eng. Biotechnol. 108, 95, Kabel et al., 2006, Bioeng. Biotechnol. 93, 56).
Therefore, when each enzyme is produced using a recombinant host system such as bacteria or yeast, it can be used in combinations optimized for the type of biomass, reducing the use of commercial enzymes. The yeast Saccharomyces cerevisiae as the host cell for the production of recombinant enzymes has superior ethanol-fermenting ability and frequently used as an ethanol-producing strain. Many studies have been made to introduce a cellulose-degrading ability into this strain and to produce recombinant cellulases (Lynd et al., 2002, Microbiol. Mol. Biol. Rev., 66, 506). However, since this strain has no cellulase productivity, its applicability in the mass-production of the recombinant cellulases is low.
As such, the yeast is very excellent in the conventional bioethanol fermentation, but it has no cellulose biomass-degrading ability. Thus, when non-food cellulosic biomass is used as a raw material for the production of bioenergy, the use of expensive cellulase is inevitably needed. In order to solve this problem, numerous studies have been conducted to develop a recombinant strain that is introduced with a foreign cellulase gene. However, its low enzyme productivity generates a problem that the cellulose substrate in the medium is not efficiently degraded by the enzymes produced. Consequently, there has been a limit in the production of bioethanol using the resulting sugar.
Accordingly, in order to solve these problems, the present inventors have prepared beta-glucosidase that is mutated to have enhanced activity, and produced a large amount of beta-glucosidase using a translational fusion partner technology which is a technology for high secretory production of the yeast recombinant protein, and they also used the recombinant enzyme-producing strain to develop an efficient CBP technology for bioethanol production based on high concentration of cellobiose and a cost-effective simultaneous saccharification and fermentation (SSF) technology for bioethanol production, thereby completing the present invention.